Molecular and phylogenetic characterization of Thelohanellus boggoti Qadri, 1962 (Cnidaria, Myxosporea, Bivalvulida) infecting the fin of Indian minor carp Labeo dero (Hamilton, 1822)

A myxozoan parasite belonging to the genus Thelohanellus Kudo, 1933 (Cnidaria, Myxosporea, Bivalvulida) was isolated from the fins of Labeo dero inhabiting Ranjit Sagar Wetland, Punjab, India. The plasmodium was 0.5-0.7 mm in diameter each containing 80- 100 number of myxospores. Myxospores were egg shaped to ovoidal in valvular view having bluntly pointed anterior and broad rounded posterior end measuring 8.36x 4.77 µm. Polar capsule was single, broadly pyriform in shape, measuring 4.77x3.98 µm in size containing a polar filament coiled perpendicular to the longitudinal axis of myxospore body making 7-9 turns. Blast analysis of 18S rDNA sequence of the isolate demonstrated 98% homogeneity with Thelohanellus sp. (KU155561, Unpublished), followed by 91% with T. caudatus (KM252684) and Thelohanellus sp. FCO (KR819273). The intensity of infection was recorded to be light as indicated by fin plasmodial index (FPI = 1). In the present study, T. boggoti has been described using 18S rRNA gene and phylogenetic analysis using MEGA.6.


INTRODUCTION
The Myxozoa are important fish parasites characterized by a two host life cycle involving invertebrates and vertebrates as definitive and intermediate hosts, respectively. There are about 2200 described myxosporean species [1] characterised by morphology and morphometrics of the myxospores representing about 18% of cnidarian species.Certain myxozoans have been recorded to cause serious damage to economically important fishes such as salmon and trout in all parts of the globe namely MBRC http://mbrc.shirazu.ac.ir 14 proliferative kidney disease caused by Tetracapsuloides bryosalmonae [2], whirling disease by Myxobolus cerebralis [3], turbot enteromyxosis by Enteromyxum scophthalmi [4] and gill disease by Thelohanellus bifurcata [5].
Ranjit Sagar Wetland, also known as the Thein Dam is at the boundary of three states of Punjab, Jammu and Kashmir and Himachal Pradesh, India on river Ravi. It is a manmade, riverine and lacustrine wetland with freshwater ecology. It lies at an altitude of about 540 msl at 32˚ 26΄ 30´´ N Latitude and 75˚ 43΄ 30´´ E Longitude and is spread over an area of 87.60 sq km. It is a cold water wetland inhabiting high diversity of fish species. Members of the genus Thelohanellus Kudo, 1933 are characterized by having shell with smooth valves and single polar capsule [6]. So far 108 nominal species have been recorded throughout the world, out of which 32 species have been described from India [7,8]. The identification of the genus is based on the spores, a propagative life cycle stage enclosed in plasmodium. The authors have been successful in characterizing some of the species of Thelohanellus from northern part of India [5,[9][10][11][12][13][14][15][16][17]. The present report is the first on the molecular and phylogenetic characterizations of T. boggoti as molecular characterizations of already described Thelohanellus species have been done by earlier workers [18,19].

MATERIALS AND METHODS
Collection and Microscopy: Specimens of Labeo dero (n= 50) with average length of 15-20 cm were procured from the various catchment sites of Ranjit Sagar Wetland. They were anesthetized with chloroform at a concentration of 40 mg/l, and necropsied to study parasites using a stereo microscope. After necropsy, plasmodia present on the fins were removed, teased on a slide and examined under phase contrast microscope (Magnus MLX) and myxospore morphology was studied, measured and photographed. The parasite frequency index (PFI) was found to be 20% (10/50). Fin plasmodial index (FPI) was calculated on the basis of number of plasmodia present per fin visible under the stereozoom binocular microscope and with the naked eye [20] using the following scale: no plasmodium (no infection-0); 1-5 plasmodia (light infection-1); 5-10 plasmodia (moderate infection-2); 10-20 plasmodia (heavy infection-3); 20-50 plasmodia or more (severe infection-4).

DNA extraction, PCR amplification and Sequencing:
The myxospores were collected from the infected fish (10 in number) and fixed in absolute alcohol for molecular and phylogenetic analysis. The parasite DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's instructions. The product was then quantified in a Nanodrop (Thermo Scientific, Wilmington, USA) spectrophotometer at 160 ng/μl. The specific primers for 18S region My1F (5'-CTA ATC CCG GTA ACG AAC GA-3') and My10R (5'-CGT CCT CGC AAC AAA CTG TA-3') standardized during the present study were used for the amplification of 18S rDNA using Eppendorf Master Cycler Pro S. The PCR was carried out, according to [21] at the final volume of 25 μl using the primers which successfully amplified the fragments of 818 bp of the 18S rDNA gene. The amplification reactions were conducted with 50 ng of genomic DNA, 12.5 μl of 1× reaction buffer (HiMedia), 1.0 μl of each primers, 1.0 μl of total DNA and 10.5 μl of nuclease free water. Amplification was done by initial denaturation at 95°C for 3 min, followed by 34 cycles of denaturation at 95°C for 30 s, annealing of primers at 57°C for 30 s, extension at 72°C for 1 min 20 s. The final extension was at 72°C for 10 min. The PCR product was analyzed on a 2% agarose gel containing 0.5 μg/ml ethidium bromide in 1× Tris-acetate-EDTA (TAE) buffer and size was estimated by comparison with the 100 bp Plus DNA Ladder. The amplified product was commercially sequenced at Molecular Diagnostic and Research Laboratories, Chandigarh (India).

Phylogenetic analysis:
The phylogenetic analysis was done on a selection of 18S rDNA sequences that comprised the new sequence (KU884967) and 25 additional sequences from closely related sequences showing 86% homogeneity or above in NCBI GenBank database using the basic local alignment tool [22]. Sphaerospora molnari (AF378345) isolated from Carassius auratus was taken as an outgroup. Genetic distance analyses were conducted using the Kimura 2-parameter model [13] in MEGA6 software [23]. Included codon positions were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. Sequence alignment was performed by Multiple Sequence Comparison by Log-Expectation (MUSCLE). The best fit substitution model for constructing the phylogenetic tree was K2+G having the lowest Bayesian score of 6481.279. The tree was generated using Neighbour-Joining having 1000 bootstrap values and was proportional to the number of substitutions per site. Tajima's neutrality values were calculated by selecting all the species. All the analysis was conducted in MEGA6. The results were exported and copied into excel files and saved as tables.

RESULTS AND DISCUSSION
Plasmodia were minute, round to irregular, creamish-white which measured 0.5-0.7 mm in diameter. They were found attached to the fins and were histozoic. There were 2-5 pasmodia per fin having 80-100 myxospores per plasmodium. Clinical signs on the fin were apparent showing pale spots (Fig. 1). Myxospores measured 8.36x4.77µm, egg shaped to ovoidal in valvular view having bluntly pointed anterior end and broad rounded posterior end. Shell valves were thick, smooth, symmetrical and measured 0.66µm in thickness. Parietal folds were absent. Polar capsule was flask-shaped with distinct neck measuring 4.77*3.98µm and occupied more than half of the myxospore body cavity. Polar filament formed 7-9 coils and was arranged perpendicular to the polar capsule axis. One capsulogenic nucleus was present beneath the polar capsule measuring 1.4µm in diameter. Sporoplasm was agranular, homogenous, moon-shaped which occupied whole of the extracapsular space behind the polar capsule. Sporoplasm contained two sporoplasmic nuclei and an iodinophilous vacuole was also present which measured 1.25x1.35µm and 1.4x1.35μm in diameter respectively (Fig. 2, Table 1).
Type   The present observations on T. boggoti Qadri, 1962 are in conformity with the original description (LS/WS: 1.70 vs 1.75) except some variations in the size of the myxospore (as indicated by LS/WS ratio) and polar capsule. Earlier, this parasite was recorded from the gills of Labeo boggot (Sykes, 1839) in Andhra Pradesh, India. Subsequently, it was recorded from the gills of Catla catla in Harike wetland, Punjab, India. In the present study, a new host-Labeo dero (Hamilton, 1822), a new locality-Ranjit Sagar wetland, Punjab, India and a new site of infection-fins have been recorded for this parasite ( Table 2). The The other clade comprised of all the Indian Thelohanelloid species from gills with a highest bootstrap value of 99. The out-group Sphaerospora molnari was phylogenetically clustered distinctly as a separate lineage (Fig. 3). Moreover, estimates of evolutionary pair-wise divergence among the sequences of T. habibpuri, Thelohanellus sp. CTPv, T. caudatus, Thelohanellus sp. FCO and Thelohanellus sp. was 0.09, 0.01, 0.08, 0.09 and 0.01 respectively (Fig. 4).
The nucleotide frequencies were 26.66% (A), 25.26% (T/U), 20.13% (C) and 27.94% (G). The transition/transversion rate ratios are K1= 3.081 (purines) and K2= 4.819 (pyrimidines). The Gamma distribution among 5 categories was 0.7349. All positions containing gaps and missing data were eliminated. Tajima's neutrality test for the nucleotide mutation was also done. The D value was found to be 1.16889 meaning some of the alleles were present at low frequencies indicating low genetic diversity among myxosporeans. The present report is the first on the molecular and phylogenetic characterizations of T. boggoti.